Development Advance Online Articles. First posted online on 29 May 2013 as 10.1242/dev.090993
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STEM CELLS AND REGENERATION
RESEARCH ARTICLE 2669
Development 140, 2669-2679 (2013) doi:10.1242/dev.090993
© 2013. Published by The Company of Biologists Ltd
Specification of hepatopancreas progenitors in zebrafish by
hnf1ba and wnt2bb
Joseph J. Lancman1,*, Natasha Zvenigorodsky2, Keith P. Gates1, Danhua Zhang1, Keely Solomon3,
Rohan K. Humphrey4, Taiyi Kuo2, Linda Setiawan2, Heather Verkade2,5, Young-In Chi6, Ulupi S. Jhala4,
Christopher V. E. Wright3, Didier Y. R. Stainier2,‡,§ and P. Duc Si Dong1,*§
SUMMARY
Although the liver and ventral pancreas are thought to arise from a common multipotent progenitor pool, it is unclear whether
these progenitors of the hepatopancreas system are specified by a common genetic mechanism. Efforts to determine the role of
Hnf1b and Wnt signaling in this crucial process have been confounded by a combination of factors, including a narrow time frame
for hepatopancreas specification, functional redundancy among Wnt ligands, and pleiotropic defects caused by either severe loss of
Wnt signaling or Hnf1b function. Using a novel hypomorphic hnf1ba zebrafish mutant that exhibits pancreas hypoplasia, as observed
in HNF1B monogenic diabetes, we show that hnf1ba plays essential roles in regulating β-cell number and pancreas specification,
distinct from its function in regulating pancreas size and liver specification, respectively. By combining Hnf1ba partial loss of function
with conditional loss of Wnt signaling, we uncover a crucial developmental window when these pathways synergize to specify the
entire ventrally derived hepatopancreas progenitor population. Furthermore, our in vivo genetic studies demonstrate that hnf1ba
generates a permissive domain for Wnt signaling activity in the foregut endoderm. Collectively, our findings provide a new model
for HNF1B function, yield insight into pancreas and β-cell development, and suggest a new mechanism for hepatopancreatic
specification.
INTRODUCTION
The liver and ventral pancreas, and presumably gall bladder and
extra hepatopancreatic ducts, are believed to develop from a
common multipotent progenitor population (hepatopancreas
progenitors) present at early somitogenesis (Deutsch et al., 2001;
Chung et al., 2008; Wandzioch and Zaret, 2009). In support of this
model, multipotency of cells within this developing organ system
appears to be maintained beyond early somitogenesis stages. For
example, in ptf1a mutants, presumptive ventral pancreas progenitors
will instead adopt intestinal (Kawaguchi et al., 2002), gall bladder,
common bile duct (Burlison et al., 2008) or liver fate (Dong et al.,
2008) (P.D.S.D., unpublished). Further observations supporting
multipotency come from Sox17, Fgf10, and Hes1 loss-of-function
studies where cells of the extra hepatopancreas ductal system can
adopt pancreas or liver fate (Sumazaki et al., 2004; Fukuda et al.,
2006; Dong et al., 2007; Spence et al., 2009). In addition, fgf10
mutants develop ectopic pancreas cells in the liver (Dong et al.,
1
Sanford Children’s Health Research Center, Programs in Genetic Disease,
Development and Aging, and Stem Cell and Regenerative Biology, Graduate School
of Biomedical Sciences, Sanford-Burnham Medical Research Institute, 10901 North
Torrey Pines Road, La Jolla, CA 92037, USA. 2Department of Biochemistry and
Biophysics, Programs in Developmental Biology, Genetics and Human Genetics,
and the Diabetes Center and Liver Center, University of California, San Francisco,
1550 Fourth Street, San Francisco, CA 94158, USA. 3Department of Cell and
Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN
37232, USA. 4Pediatric Diabetes Research Center, UCSD School of Medicine, La Jolla
CA 92037, USA. 5School of Biological Sciences, Monash University, Clayton, VIC
3800, Australia. 6Hormel Institute, University of Minnesota, Austin, MN 55912, USA.
*These authors contributed equally to this work.
‡
Present address: Department of Developmental Genetics, Max Planck Institute for
Heart and Lung Research, 61231 Bad Nauheim, Germany
§
Authors for correspondence (didier.stainier@mpi-bn.mpg.de;
ducdong@sanfordburnham.org)
Accepted 25 April 2013
2007), and both fgf10 and sox9 mutants develop liver cells in the
pancreas (Dong et al., 2007; Seymour et al., 2012). These examples
demonstrating that cells within the developing hepatopancreas
organ system can readily switch fate is consistent with the liver,
ventral pancreas, gall bladder and associated ducts arising from a
common multipotent progenitor pool.
It remains unresolved whether a common genetic mechanism
specifies this entire progenitor population. Although significant
progress has been made in identifying factors necessary for either
pancreas or liver specification, factors uniquely required for
induction of the entire ventrally derived hepatopancreas system
have yet to be discovered. Several transcription factors including
Ptf1a and Pdx1 have been implicated in ventral pancreas
specification, but neither factor is required for liver specification
(Offield et al., 1996; Kawaguchi et al., 2002). In Hhex or Gata4
mutants, the ventral pancreas is not specified, but early liver markers
are expressed, suggesting liver specification has occurred (Bort et
al., 2004; Bort et al., 2006; Watt et al., 2007). Although Foxa1 and
Foxa2 function redundantly to establish endoderm competence
necessary for liver specification (Kaestner, 2005; Lee et al., 2005),
whether these factors act broadly to affect foregut endoderm
patterning or have a more restricted role in liver and ventral
pancreas specification has not been addressed .
Zebrafish and mouse embryos with severe or complete loss of
HNF1B (also known as HNF1β, TCF2 and VHNF1) function
exhibit profound foregut regionalization defects and subsequently
fail to specify the liver and both the dorsal and ventral pancreas (Sun
and Hopkins, 2001; Haumaitre et al., 2005; Lokmane et al., 2008).
For this reason, it is unresolved whether the liver or pancreas
agenesis defects observed in severe hnf1b loss-of-function mutants
are secondary to the earlier foregut patterning defects. Loss of
wnt2bb in zebrafish can lead to a low penetrant liver specification
defect. Although the penetrance of this defect is increased when
DEVELOPMENT
KEY WORDS: Hnf1b, Pancreas, Liver, Wnt, Diabetes, MODY
Development 140 (13)
2670 RESEARCH ARTICLE
from a synergistic genetic interaction between hnf1ba and wnt2bb,
we uncover a new mechanism for hepatopancreas progenitor
specification, revealing that a common genetic program, within a
narrow developmental time window, specifies progenitors of the
liver, ventral pancreas, gall bladder and associated ducts.
MATERIALS AND METHODS
Zebrafish strains
Zebrafish were raised and maintained under standard laboratory
conditions. We used the following mutant and transgenic lines: ligers430
(hnf1bas430), hnf1bahi2169 (Sun and Hopkins, 2001), wnt2bbs404 (Ober
et al., 2006), Tg(hsp70l:wnt8-GFP)w34 (Weidinger et al., 2005),
Tg(elastase:GFP);Tg(lfabp:dsRed)gz2 (Wan et al., 2006), Tg(hsp70l:dkk1GFP)w32 (Stoick-Cooper et al., 2007) and Tg(ptf1a:eGFP)jh1 (Godinho et
al., 2005).
Genotyping of mutant embryos
hnf1bas430 genotyping
Genomic DNA was amplified with (F:5⬘-AGCAGCACAATATCCCTCAGCGCGAGGTCG-3⬘) and (R:5⬘-AAAACCGTCACATGCAACAA3⬘) primers. Amplicons were then digested overnight with Taqα1 (NEB).
The forward primer introduces a mismatch creating a Taqα1 restriction site
in the hnf1bas430 amplicon (Neff et al., 1998). Following digestion, the wildtype allele generates a band of 195 bp and the ligers430 allele a band of 165
bp.
wnt2bbs404 genotyping
Genomic DNA was amplified with (F:5⬘-CGTTCGTATACGCGATCTCC3⬘) and (R:5⬘-TTCCACAGCGGTTGTTATGA-3⬘) primers followed by
digestion with Fok1. The wild-type allele generates a band of 258 bp and the
wnt2bbs404 allele a band of 152 bp. hnf1bahi2169 mutants were genotyped as
described previously (Sun and Hopkins, 2001).
Morpholino injections
One-cell stage embryos were injected with varying amounts of a hnf1ba
morpholino (0.28-1.7 ng) (5⬘-GGGAAATGCGGTATTGTGATCTTTC-3⬘)
(Gene Tools).
Heat-shock conditions
Embryos were heat-shocked at 21 hpf by adding egg-water pre-warmed to
38°C for Tg(hsp70l:dkk1-GFP)w32 and 39°C for Tg(hsp70l:wnt8-GFP)w34
and maintained at 37°C and 38°C, respectively, for 7 hours. Following heatshock, embryos were sorted, returned to 27.5°C and collected at specified
stages.
Fluorescent immunohistochemistry
Whole-mount fluorescent immunohistochemistry was performed as
previously described (Dong et al., 2007), using the following antibodies:
mouse anti-Islet1/2 [1:10; Developmental Studies Hybridoma Bank
(DSHB)], rabbit anti-pan-Cadherin (1:5000; Sigma), guinea pig anti-Insulin
(1:500; Biomeda), mouse anti-Synaptic Vesicles (SV2; 1:20; DSHB),
guinea pig anti-Pdx1 (1:200), rabbit anti-Prox-1 (1:200; Millipore) and
mouse anti-Myosin to label somites following in situ hybridization (1:20;
DSHB, F59), fluorescently conjugated Alexa secondary antibodies
(Molecular Probes) and DAPI. Control and mutant (or morphant) embryos
were stained in the same tube for each experiment. To analyze β-cell
numbers, day 3 and 5 hnf1bs430 and wild-type embryos were processed for
confocal imaging using DAPI and antibodies against insulin and synaptic
vesicles. Z-stacks (0.8 μm steps) were collected for the entire islet with
optical slices captured at a focal depth of 1.6 μm. Samples were imaged
using a Zeiss LSM5 Pascal or Zeiss 710 confocal microscope.
Reporter constructs and assay
Full-length zebrafish wild-type and mutant hnf1ba (V147E) were subcloned
into pcDNA3-Myc vector and co-transfected with β28-Luciferase reporter
(a kind gift from Dr G. Crabtree, Stanford University, CA, USA) containing
three copies of the HNF1-binding site from the fibrinogen promoter and
with RSV-β-Gal, into Min6 or HepG2 cells in the absence or presence of
both CMV Flag-pCAF or CMV Flag-CBP expression vectors, as indicated.
DEVELOPMENT
combined with wnt2 loss of function, swimbladder agenesis is also
observed and, importantly, ventral pancreas specification still occurs
(Ober et al., 2006; Poulain and Ober, 2011). It is not known whether
other Wnt ligands act redundantly with wnt2bb and wnt2 to specify
the ventral pancreas. Nevertheless, Wnt signaling has been
implicated in pancreas development. Studies in frog showed that
Wnt signaling before early somitogenesis plays a negative role in
liver and pancreas development by inhibiting foregut identity
(McLin et al., 2007). Studies in mice suggest that ectopic Wnt
signaling during early pancreas development can antagonize
pancreas specification (Heller et al., 2002; Heiser et al., 2006;
Murtaugh, 2008). However, between these developmental stages,
whether endogenous Wnt signaling regulates ventral pancreas
specification has yet to be determined.
HNF1B is the gene implicated in maturity onset diabetes of the
young 5 (MODY5), one of ten monogenic, dominantly inherited
forms of early-onset diabetes (Horikawa et al., 1997; Borowiec et
al., 2009; Nyunt et al., 2009; Wang et al., 2009). Based on genomewide association (GWA) studies, HNF1B and its closely related
paralog HNF1A (MODY3), are also associated with type 2 diabetes
(T2D), suggesting broader significance for studying these
monogenic diabetes genes (Frayling, 2007; Wang et al., 2009;
Voight et al., 2010). Loss of insulin producing pancreatic β-cells is
characteristic of nearly all forms of diabetes and is the underlying
factor for diabetic insulin dependence. While it has been suggested
that HNF1B is important for liver insulin sensitivity, it remains
unclear whether its dysfunction in the pancreas, particularly the βcells, contributes to the etiology of HNF1B-related diabetes
(Brackenridge et al., 2006; Kornfeld et al., 2013). Although targeted
knockout of Hnf1b from β-cells in mice leads to impaired glucose
tolerance, fed or fasted plasma glucose and insulin levels were not
affected, suggesting that Hnf1b may not be crucial for maintaining
β-cell function (Wang et al., 2004). However, it is not known
whether Hnf1b is required for normal β-cell development. In
support of a role in β-cell development, lineage-tracing studies in
mice reveal that Hnf1b is expressed in pancreas endocrine
progenitors that can give rise to β-cells during development (Solar
et al., 2009). GWA studies have also implicated the Wnt pathway,
including WNT2B, as the highest ranked pathway associated with
T2D (Perry et al., 2009). By analyzing the role of wnt2bb and hnf1b
in pancreas development, we aim to advance our understanding of
β-cell development and diabetes etiology.
The heterozygous nature of the HNF1B mutations in individuals
with MODY5 suggests that partial loss of HNF1B function can lead
to disease pathologies. However, to date only complete or severe
loss-of-function Hnf1b alleles have been examined in mouse and
zebrafish (Barbacci et al., 1999; Sun and Hopkins, 2001; Haumaitre
et al., 2005; Lokmane et al., 2008). As would be expected, Hnf1bnull mutants exhibit more pleiotropic and severe defects than those
observed in individuals with MODY5, limiting their utility as
development and disease models. Whether HNF1B plays a distinct
role in β-cell development or in pancreas and liver specification has
been difficult to study because of the broader endoderm patterning
defects associated with its severe loss of function. Our studies
provide new insights into HNF1B function by uncoupling these
defects. We report here a novel hypomorphic hnf1ba zebrafish
mutant that does not exhibit significant foregut endoderm
regionalization defects. Our analysis of this mutant leads to several
important advances. We find that partial loss of hnf1ba function in
zebrafish can cause MODY5-like pancreas defects. We also find
that normal levels of hnf1ba function are distinctly required for
proper β-cell numbers and for pancreas specification. Furthermore,
Luciferase was measured using a luminometer, and values normalized for
expression of β-Gal as described.
In situ hybridization
In situ hybridization was performed as described with minor modifications
(Yelon et al., 1999; Dong et al., 2007). Following in situ hybridization,
immunohistochemistry was performed to fluorescently label somites as
previously described (Dong et al., 2007; Huang et al., 2008). Images were
captured using a Zeiss 710 confocal microscope with AxioPlan 4.8 software.
In situ hybridization and immunofluorescent images were captured for a
single sample and merged using Photoshop CS3. Probes for hnf1ba, hhex,
prox1, cmyc (myca – Zebrafish Information Network) and wnt2bb were
donated and are described elsewhere (Ho et al., 1999; Sun and Hopkins,
2001; Yamaguchi et al., 2005; Ober et al., 2006; Shin et al., 2011).
Experimental and control embryos were stained in the same tube and all
experiments were carried out at least twice using distinct clutches.
RESULTS
Identification and molecular characterization of a
new hnf1ba hypomorphic zebrafish mutant
From a zebrafish ENU mutagenesis screen (Ober et al., 2006), we
identified ligers430, a recessive mutant that exhibits a variably
smaller pancreas, as marked by Tg(ptf1a:eGFP)jh1 expression
(Godinho et al., 2005; Dong et al., 2008) (Fig. 1A,B, arrows indicate
pancreas). ptf1a, which encodes a basic helix-loop-helix
transcription factor, is expressed in the pancreatic anlagen and is
required for ventral pancreas specification (Kawaguchi et al., 2002;
Lin et al., 2004; Zecchin et al., 2004; Dong et al., 2008). The
ligers430 mutation is linked to the simple sequence length
polymorphism (SSLP) marker z4396 on chromosome 15, in the
same region as hnf1ba (zfin.org).
Complementation analysis with a severe hnf1ba mutant,
hnf1bahi2169, yielded transheterozygotes that phenocopied the
ligers430 homozygous variable pancreas hypoplasia (with greater
severity and penetrance), indicating that the ligers430 mutation is a
hypomorphic allele of hnf1ba (Fig. 1C-F). In addition, ligers430
homozygous and ligers430/hnf1bahi2169 transheterozygous embryos
do not exhibit kidney cysts (not shown) or liver agenesis, as reported
in the hnf1bahi2169, hnf1bahi548, hnf1bahi1843, hnf1bawi408 and
RESEARCH ARTICLE 2671
hnf1bala550 homozygotes, providing evidence that ligers430 is
hypomorphic to these published alleles (Sun and Hopkins, 2001;
Wiellette and Sive, 2003; Song et al., 2007). Morpholino (MO)
antisense knock-down of hnf1ba translation in embryos (morphants)
phenocopies the range of pancreas hypoplasia and agenesis defects
observed in ligers430 homozygotes and ligers430/hnf1bahi2169
transheterozygotes, depending on the amount injected (Fig. 1G-I;
supplementary material Fig. S3), providing further evidence that
the ligers430 mutation is a hypomorphic allele.
Sequence analysis of hnf1ba in ligers430 mutants revealed a
transversion of a thymine to an adenine in the coding region at
position 440, resulting in a valine to glutamic acid (V147E)
missense mutation (Fig. 1J). This valine is highly conserved among
vertebrates and is in the DNA-binding POU-specific domain where
MODY5 and MODY3 (Hnf1a) missense mutations are most
commonly found (Fig. 1K) (Chi et al., 2002; Gong et al., 2004;
Edghill et al., 2006a). Based on crystal structure data of human
HNF1B in complex with DNA, V140 in human HNF1B, which is
homologous to V147 in zebrafish (Fig. 1K), is predicted to be
involved in the formation of the POU-specific domain hydrophobic
core but not make direct contact with DNA (supplementary material
Fig. S1A) (Lu et al., 2007). Therefore, substitution of a highly
charged group (V147E) is predicted to perturb the hydrophobic core
and disrupt protein structure and function.
Consistent with the predicted structural model, we find that when
compared with wild-type zebrafish Hnf1ba, V147E Hnf1ba fails to
efficiently activate transcription of a luciferase reporter driven by a
multimerized HNF1-binding element of the fibrinogen gene
(supplementary material Fig. S1B) (Chi et al., 2002). In addition,
although decreased relative to wild-type Hnf1ba, V147E Hnf1ba
transcriptional potential can be enhanced by known co-activators
pCAF and CBP (Barbacci et al., 2004), further supporting that
V147E Hnf1ba retains some function (supplementary material Fig.
S1B). Together with genetic linkage to the hnf1ba locus, phenocopy
of ligers430 homozygous phenotypes by hnf1ba knock-down, failure
of complementation by hnf1bahi2169 and substitution of a highly
conserved residue of Hnf1ba, we conclude that ligers430 (now
designated hnf1bas430) is caused by a hypomorphic mutation in
Fig. 1. ligers430 is a hypomorphic hnf1ba zebrafish mutant
and exhibits MODY5-like pancreas hypoplasia.
(A,B) Merged fluorescent/bright-field micrographs of 4 dpf
Tg(ptf1a:eGFP)jh1 (arrows indicate pancreas) wild type (A, top)
and ligers430 mutants showing the most common severity of
pancreas hypoplasia (A, bottom). In ligers430 mutant embryos,
the pancreas can be either nearly normal in size (B, top; <10%)
or completely absent (B, bottom; <10%). (C-I) Threedimensional rendering of the foregut endoderm of 80 hpf
Tg(ptf1a:eGFP)jh1 wild-type (C), ligers430 (D,E), ligers430/
hnf1bahi2169 (F) and hnf1ba-MO injected (G-I) embryos stained
for Isl1 and cadherin demonstrating complementation failure
by hnf1bahi2169 (F) and phenocopy with Hnf1ba translational
knock-down (compare D-F with G-I, respectively). (J) Genomic
sequence from a ligers430 heterozygote indicating a molecular
lesion (arrow; double peek) in hnf1ba. (K) Amino acid
alignment covering part of the atypical POU-specific domain
of human HNF1A and HNF1B and wild-type zebrafish and
ligers430 Hnf1ba. Red font denotes the valine to glutamic acid
substitution at position 147 in ligers430. Blue font indicates
amino acids affected by mis-sense mutations in MODY3 and
MODY5.
DEVELOPMENT
Hepatopancreas specification
2672 RESEARCH ARTICLE
Development 140 (13)
hnf1bas430 hypomorphs exhibit a MODY5-like
pancreas hypoplasia and have reduced β-cell
numbers
Foregut regionalization defects and pancreas agenesis in severe
Hnf1b loss-of-function zebrafish and mouse mutants limit their
utility as models for Hnf1b function in the developing and adult
pancreas (Sun and Hopkins, 2001; Haumaitre et al., 2005). With
this newly identified hypomorphic hnf1ba mutant, we have the
unique opportunity to examine the consequence of Hnf1ba partial
loss of function during pancreatogenesis. We find that the
developing hnf1bas430 mutant exocrine pancreas, which is thought
to arise exclusively from the ventral pancreas in zebrafish (Field et
al., 2003), is variably reduced in size based on Tg(ptf1a:eGFP)jh1
expression (Fig. 1A,B). In fewer than 15% of hnf1bas430
homozygotes (n>300), the ventral pancreas can appear either
normal in size (<10%) or completely absent (<10%), with variation
between clutches. Because we find that MODY5-like pancreas
hypoplasia can result from a quantitative loss of Hnf1ba (via
translational knock-down), we propose that pancreas hypoplasia in
MODY5 is due to decreased levels of HNF1B function. The dorsal
pancreas, which comprises primarily endocrine cells in zebrafish
(Field et al., 2003; Hesselson et al., 2009), is dysmorphic in
hnf1bas430 homozygotes. In mutant embryos, endocrine cells, which
arise from both the dorsal and ventral pancreas, can fail to form a
single islet cluster. Furthermore, the clusters of endocrine cells that
do form do not organize into an islet with a central core of β-cells,
normally observed in zebrafish and mice. We find a significant and
consistent reduction in the number of β-cells in hnf1bas430
homozygotes, regardless of exocrine size, although the severity of
reduction generally correlates with exocrine size (Fig. 2A). To
determine whether the roles of hnf1ba in regulating β-cell number
and exocrine size are distinct, we compared β-cell numbers in 80 hpf
wild-type embryos with those of 125 hpf mutants with a similar or
larger exocrine pancreas. We find that age-matched or older mutant
embryos with an exocrine pancreas similar to or larger than that of
an 80 hpf wild type have significantly fewer β-cell numbers (n>20)
(Fig. 2B-G; supplementary material Fig. S2A-D). This finding
indicates that hnf1ba regulation of β-cell number is distinct from its
regulation of pancreas size.
hnf1ba plays a distinct role in ventral pancreas
specification
Although severe loss of hnf1ba does lead to ventral pancreas
agenesis in zebrafish, it also causes loss of adjacent endodermal
organs, including the liver, dorsal pancreas and swim bladder,
indicating a more profound role for hnf1ba in posterior foregut
patterning (Sun and Hopkins, 2001). In Hnf1b-null mice, in which
ventral pancreas agenesis occurs, only rudiments of nondifferentiating liver and dorsal pancreas buds were observed, also
suggesting a broader role for Hnf1b in endoderm development and
potentially obscuring a distinct role in pancreas specification
(Haumaitre et al., 2005). Consistent with a role for Hnf1b in AP
(anterior/posterior) patterning of the foregut endoderm, both
Fig. 2. Distinct roles for Hnf1ba in regulating β-cell numbers and
ventral pancreas specification. (A) β-Cell nuclei numbers from 80 and
125 hpf wild-type and hnf1bas430 mutants, indicating that at either stage,
hnf1bas430 mutants have significantly fewer β-cells. hnf1bas430 mutants
(125 hpf ) have significantly fewer β-cells than 80 hpf wild-type embryos.
Error bars represent s.d. (B-G) Fluorescent confocal microscopy of
Tg(ptf1a:eGFP)jh1 80 hpf wild type (B-D) and 125 hpf hnf1bas430 mutant (EG) pancreas stained for insulin antibodies, SV2 antibodies and DAPI to
mark β-cells (red), endocrine cells (white) and nuclei (blue), respectively.
Three-dimensional rendering of red and green channels showing an
hnf1bas430 mutant at 125 hpf with a larger pancreas (E) than wild-type (B).
(C,F) Magnification (8×) of B and E (red and white channels only) to show
mildly disorganized islet cells. (D,G) Z-focal plane of C and F (with all
channels) demonstrating moderately reduced β-cell number in the
hnf1bas430 mutant islet. For a more severe example, see supplementary
material Fig. S2. (H-M) Three-dimensional rendering of 52 hpf
Tg(ptf1a:eGFP)jh1 (pancreas, green) foregut endoderm in wild-type (H),
hnf1bahi2169 mutants (I) and hnf1bas430 embryos (J) stained for Pdx1 to
mark the duodenal intestine and cadherin to mark the endoderm
epithelium. In contrast to hnf1bahi2169 (I), Pdx1 expression (arrowheads) in
the intestine and dorsal pancreatic islet (I) is not lost in hnf1bas430
embryos (J). (K-M) Three-dimensional rendering of 80 hpf
Tg(ptf1a:eGFP)jh1; Tg(lfabp:dsRed)gz2 (pancreas, green; liver, red) foregut
endoderm in wild-type (K) hnf1bahi2169 (L) and hnf1bas430 embryos (M)
stained for cadherin showing specific loss of ventral pancreas in
hnf1bas430 hypomorphic mutants. Unlike the hnf1bahi2169 (L), liver (L) and
swimbladder (SB) are not lost in the hnf1bas430 embryos.
DEVELOPMENT
hnf1ba. We note that hnf1bb, a paralog of hnf1ba in zebrafish, was
not reported to be expressed in the developing foregut endoderm
(Choe et al., 2008; Wingert and Davidson, 2011; Naylor et al.,
2013). Because loss of hnf1ba in zebrafish and Hnf1b in mouse
exhibit similar foregut endoderm phenotypes (Sun and Hopkins,
2001; Haumaitre et al., 2005; Lokmane et al., 2008), we suggest
that hnf1ba is the primary functional ortholog of mammalian Hnf1b
in foregut development.
zebrafish and mouse Hnf1b-null mutants exhibit a posterior
expansion of hnf3b and sonic hedgehog (shh) expression into the
presumptive duodenum (posterior foregut), the region from which
the ventral and dorsal pancreas and liver develop (Sun and Hopkins,
2001; Haumaitre et al., 2005). Hedgehog signaling in the anterior
endoderm is required early to pattern the anterior foregut via
repression of more posterior endoderm fate (duodenum/pancreas/
liver) (Hebrok, 2003; Wan et al., 2006). Therefore, posterior
expansion of shh expression would be expected to inhibit posterior
foregut patterning events. Accordingly, zebrafish and mouse hnf1bnull embryos exhibit loss of expression of the posterior foregut
endoderm (pancreas/duodenum) marker, pdx1, but not loss of the
anterior foregut/endoderm markers hnf3b, nkx2.1 and irx2 (Sun and
Hopkins, 2001; Haumaitre et al., 2005; Lokmane et al., 2008).
These observations indicate a conserved effect of severe hnf1b lossof-function mutations on the AP patterning of the vertebrate foregut
(Sun and Hopkins, 2001; Haumaitre et al., 2005). In contrast to
hnf1bahi2169 mutants, AP foregut endoderm patterning appears
normal in hypomorphic hnf1bas430 embryos with pancreas agenesis.
Duodenal Pdx1 expression is not lost in these hnf1bas430 mutants,
suggesting that the duodenal region (posterior foregut) is properly
patterned (Fig. 2J). More important, adjacent endodermal organs
(swim bladder, dorsal pancreas and liver) do develop and
differentiate in hnf1bas430 mutants with ventral pancreas agenesis,
further indicating that foregut AP patterning is not significantly
impaired (Fig. 2K-M). These results reveal that ventral pancreas
RESEARCH ARTICLE 2673
development is more sensitive to reduced Hnf1ba function than
neighboring tissues, thereby suggesting that hnf1ba is distinctly
required for ventral pancreas specification, independent of its role
in foregut endoderm regionalization and liver specification. Our in
vivo data are consistent with previous molecular studies suggesting
that Hnf1b transcriptionally activates Hnf6 to regulate Pdx1 and
ventral pancreas specification (Poll et al., 2006).
Synergistic genetic interaction between hnf1ba
and wnt2bb reveals their novel role in
hepatopancreas specification
It is unresolved whether hnf1b also plays a distinct role in liver
specification because of the difficulty in uncoupling the liver
agenesis phenotype from the foregut regionalization defects
exhibited by severe hnf1b mutants (Sun and Hopkins, 2001;
Lokmane et al., 2008). Because wnt2bb mutants display rare liver
specification (<1%, n=200) and transient hypoplasia defects (Ober
et al., 2006) (Fig. 3A,B), and because hnf1bas430 mutants exhibit
variable but mild hepatic hypoplasia (Fig. 3A,C), we tested for a
synergistic genetic interaction between hnf1bas430 and wnt2bbs404
with respect to liver specification. Remarkably, about 80% of the
wnt2bbs404; hnf1bas430 double mutants (44/55) completely lack
hepatocytes, as assessed by Tg(lfabp:dsRed)gz2 expression,
indicating that hnf1ba is indeed required for liver specification. The
penetrance and severity of the liver agenesis and hypoplasia defects
in double mutants is significantly greater than the additive
Fig. 3. hnf1ba and wnt2bb cooperate to specify the hepatopancreas organ system after mid-somitogenesis. (A-F) Three-dimensional rendering
of 80 hpf Tg(ptf1a:eGFP)jh1; Tg(lfabp:dsRed)gz2 (pancreas, green; liver, red) foregut endoderm in wild type (A), wnt2bbs404 mutant (B), hnf1bas430 mutant (C)
and wnt2bbs404; hnf1bas430 double mutants (D-F) stained for cadherin (epithelial endoderm) and with monoclonal antibody 2F11 to mark extra
hepatopancreas ducts (*) (dashed outline). Relative to wild type, the wnt2bbs404 mutant liver (L) and the hnf1bas430 mutant pancreas (P) are reduced in
size. The wnt2bbs404; hnf1bas430 double mutant liver and pancreas (arrow) are more severely reduced, or lost. Double mutants can exhibit complete loss
of the hepatopancreas system (E,F; white arrowheads). (G,H) Three-dimensional rendering of 80 hpf Tg(ptf1a:eGFP)jh1 (pancreas, green) foregut
endoderm in wild-type (G) and wnt2bbs404; hnf1bas430 double mutants (H) stained for cadherin and Isl1 to mark the hepatopancreas mesenchyme
(brackets) and islet (I). Double mutants lacking the entire hepatopancreas system still develop Isl1+ mesenchyme and islet (yellow arrowheads indicate
the swimbladder). (I) Merged fluorescent/brightfield micrographs of 4 dpf Tg(ptf1a:eGFP)jh1; Tg(lfabp:dsRed)gz2 wnt2bbs404 mutant (top) and wnt2bbs404;
hnf1bas430 double mutants (bottom) showing no obvious body phenotypes other than liver and pancreas agenesis (arrows). (J,K) Three-dimensional
rendering of 80 hpf foregut endoderm, heat-shock-induced Tg(hsp70l:dkk1-GFP)w32; Tg(ptf1a:eGFP)jh1; Tg(lfabp:dsRed)gz2 and Tg(hsp70l:dkk1-GFP)w32;
hnf1bas430; Tg(ptf1a:eGFP)jh1; Tg(lfabp:dsRed)gz2 stained for cadherin and with 2F11. Heat-shock-induced expression of dkk1-GFP in wild-type background
does not lead to liver or pancreas agenesis (J), whereas in the hnf1bas430 mutant background, the hepatopancreas system is completely lost (K) (white
arrow). The swimbladder (yellow arrowhead) is not lost in wnt2bbs404; hnf1bas430 double mutants or in heat-shock-induced Tg(hsp70l:dkk1-GFP)w32;
hnf1bas430 mutants (D-F,H,K).
DEVELOPMENT
Hepatopancreas specification
penetrance and severity of the single mutants, indicating a
synergistic genetic interaction. Surprisingly, pancreatic agenesis/
hypoplasia is also more severe and penetrant in double mutants
(Fig. 3D), revealing a novel role for wnt2bb signaling in pancreas
specification (wnt2bbs404 mutants do not exhibit obvious pancreas
hypoplasia). Strikingly, in 6% of the double mutants (3/48), the
entire ventrally derived hepatopancreas system, including the
pancreas, gallbladder, extra-hepatopancreas ducts [assessed with
2F11 antibodies (Crosnier et al., 2005; Dong et al., 2007)] and liver,
is absent (Fig. 3E,F), suggesting a more profound role for hnf1ba
and wnt2bb in specifying these developmentally associated organs
(supplementary material Table S1). Islet1 (Isl1) expression in
mesenchymal cells that surround the extra-hepatopancreas ducts
and adjacent intestine (Dong et al., 2007) appears normal in these
double mutants (Fig. 3G,H). Importantly, neighboring endodermal
organs, including the swim bladder and dorsal pancreas, do develop
in these double mutants, indicating that foregut AP patterning is not
significantly affected in the wnt2bbs404; hnf1bas430 double mutants.
Other than hepatopancreas organ system defects, wnt2bbs404;
hnf1bas430 double homozygous embryos are indistinguishable from
their wild-type and single mutant siblings, based on brightfield
microscopy (Fig. 3I; Fig. 1A,B). Therefore, agenesis defects
observed in the wnt2bbs404; hnf1bas430 double mutants are highly
restricted to ventrally derived hepatopancreas organs. Furthermore,
unlike wnt2bbs404 or hnf1bas430 single mutants, which can be
homozygous viable as adults, double mutants do not survive beyond
9 dpf, indicating a synthetic lethal genetic interaction. To our
knowledge, this is the first report of a genetic condition that leads
to specific and complete agenesis of the entire ventrally derived
hepatopancreas system, demonstrating that the liver and ventral
pancreas are specified by a common genetic mechanism.
Wnt signaling interacts with hnf1ba after midsomitogenesis to specify hepatopancreas
progenitors
Current models suggest that genetic events regulating liver and
pancreas specification occur during early somitogenesis (Chung et
al., 2008; Wandzioch and Zaret, 2009; Shin et al., 2011). However,
the liver and ventral pancreas do not bud until after midsomitogenesis, suggesting that other genetic events may more
Development 140 (13)
directly regulate hepatopancreas fate in the foregut endoderm. To
assess the temporal requirement of the synergistic genetic
interaction between Wnt signaling and Hnf1ba in hepatopancreas
specification, we mis-expressed the Wnt/β-catenin antagonist
Dickkopf (Dkk1) (Glinka et al., 1998) in a temporally specific
manner via heat-shock using the Tg(hsp70l:dkk1-GFP)w32 line in
the hnf1bas430 background (Fig. 3J,K). Heat-shock of
Tg(hsp70l:dkk1-GFP)w32 or hnf1bas430 embryos for 7 hours
beginning at 21 hours post fertilization (hpf, ~24-somite stage,
3 hours before initial expression of the hepatopancreas progenitor
markers hhex and prox1) did not yield hepatopancreas agenesis
phenotypes (Fig. 3J; not shown). However under the same
conditions, 38% (5/13) of the Tg(hsp70l:dkk1-GFP)w32 embryos in
the hnf1bas430 mutant background showed a loss of the entire
hepatopancreas system, including the liver, ventral pancreas, and
associated ducts (Fig. 3K). These hepatopancreas phenotypes were
indistinguishable from those of wnt2bbs404; hnf1bas430 double
mutants, though they occurred with greater penetrance. Importantly,
we emphasize that all organ agenesis defects observed are
permanent and do not recover at later stages, strongly supporting a
specification defect rather than a general developmental delay. The
complete loss of the entire hepatopancreas organ system in
wnt2bbs404; hnf1bas430 double mutants and Tg(hsp70l:dkk1GFP)w32; hnf1bas430 embryos suggests a possible defect in
specification of hepatopancreas progenitors. Consistently, heatshocked Tg(hsp70l:dkk1-GFP)w32; hnf1bas430 embryos lack early
Prox1 and hhex expression in the ventral foregut at 36 hpf (a stage
when their expression in both liver and pancreas progenitors are
consistently detectable in wild type and single mutants) without
detectable reduction of Pdx1 expression in the presumptive
duodenum, indicating a specific loss of hepatopancreas progenitors
(Fig. 4A-H; n=30). Interestingly, prox1 and hhex, which we
demonstrated to be downstream of Hnf1ba and Wnt signaling
during hepatopancreas specification, have also been associated with
increased diabetes risk by GWA studies (Dupuis et al., 2010). A
genetic interaction among these factors in hepatopancreas
development may hint at their potential interactions in contributing
to diabetes. We note that heat-shock induction at later stages (23
hpf or later) dramatically decreased the penetrance of the
hepatopancreas system agenesis phenotype, suggesting that initial
Fig. 4. Specification of hepatopancreas progenitors by Hnf1ba and Wnt signaling. (A-D) 3D rendering (lateral view showing somites for positional
reference) of 36 hpf foregut endoderm in wild-type (A), Tg(hsp70l:dkk1-GFP)w32 (B), hnf1bas430 (C) and Tg(hsp70l:dkk1-GFP)w32; hnf1bas430 embryos (D)
stained for Prox1, Pdx1 and myosin (somites). Relative to wild type, Prox1 foregut expression (arrowheads) is similar or mildly reduced in both
Tg(hsp70l:dkk1-GFP)w32 and hnf1bas430 embryos. Prox1 expression is severely reduced or lost in Tg(hsp70l:dkk1-GFP)w32; hnf1bas430 embryos. (E-G) Double
in situ hybridization and antibody staining (ventral view) for the early hepatopancreas progenitor marker hhex and myosin at 36 hpf. hhex is expressed
(arrowheads) in the foregut endoderm and dorsal pancreas (dp) in wild type (E) but reduced in both the Tg(hsp70l:dkk1-GFP)w32 (F) and the hnf1bas430
(G) embryos. (H) hhex expression (arrowhead) is undetectable in the foregut endoderm of Tg(hsp70l:dkk1-GFP)w32; hnf1bas430 embryos.
DEVELOPMENT
2674 RESEARCH ARTICLE
Wnt signaling between 21 and 23 hpf is crucial for hepatopancreas
specification. And based on when Wnt inhibition was needed to
efficiently block hepatopancreas specification, we suggest that
between 21 and 28 hpf, just prior to liver budding, Wnt signaling is
required for hepatopancreas progenitor specification. Blocking Wnt
signaling at later stages (48 hpf) via ectopic dkk1 expression led to
a reduction in liver size (Goessling et al., 2008), suggesting a later
role for Wnt signaling in liver expansion, distinct from its earlier
role in hepatopancreas progenitor specification.
hnf1ba is necessary for Wnt signaling activity in
the foregut endoderm
The highly specific loss of the hepatopancreas system in wnt2bbs404;
hnf1bas430 double mutants suggests that Hnf1ba and Wnt2bb
functions converge at the foregut endoderm to induce
hepatopancreas fate. To determine the mechanism of this genetic
interaction, we analyzed the expression of hnf1ba, wnt2bb and
prox1 at the earliest stage of hepatopancreas development (Sun and
Hopkins, 2001; Ober et al., 2006; Huang et al., 2008; Lokmane et
al., 2008; Noël et al., 2008). At 24 hpf, hnf1ba expression extends
from the presumptive pancreas/liver to the presumptive duodenal
regions of the foregut endoderm (from AP region of somite 1-5;
Fig. 5A-C). At this stage, wnt2bb shows confined expression
(somite 1-2) in the lateral plate mesoderm adjacent to the anteriormost region of the hnf1ba expression domain. Coincidently, it is in
this anterior subdomain of hnf1ba expression that initial prox1
expression becomes detectable. Together with our functional data,
we hypothesize that hnf1ba expression defines the area of the
RESEARCH ARTICLE 2675
foregut endoderm that can be induced by Wnt signaling to express
Prox1 and adopt hepatopancreas fate (Fig. 5D). According to this
model, broad overexpression of Wnt ligands should lead to posterior
expansion of Prox1 expression where hnf1ba is expressed. As
predicted and consistent with previous studies (Poulain and Ober,
2011; Shin et al., 2011), broad overexpression of Wnt8a via heatshock of Tg(hsp70l:wnt8a)w34 embryos (Weidinger et al., 2005)
beginning at 21 hpf resulted in a robust posterior expansion of Prox1
expression within the hnf1ba-positive domain of the foregut
endoderm (Fig. 5E,F; n=30). Crucially, in hnf1bas430/hi2169
transheterozygotes, Prox1 expression is either not induced or is
weakly induced in the foregut endoderm following ectopic Wnt8
expression (Fig. 5G; 100%, n=6; and inconsistently induced in
hnf1bs430 homozygotes; not shown). These data demonstrate that
hnf1ba function is indeed required for Wnt signaling to induce
hepatopancreas fate. A possible mechanism for this genetic
interaction is that hnf1ba is necessary for transduction of the Wnt
signal in the foregut endoderm. To investigate this, we examined
the expression of cmyc (myca), a direct Wnt/β-catenin target (He et
al., 1998), which is expressed in hepatopancreas progenitors and
whose expression domain can be induced posteriorly by ectopic
Wnt8a expression (Fig. 5H) (Yamaguchi et al., 2005; Shin et al.,
2011). Intriguingly, this posterior expansion also correlates with the
hnf1ba expression domain (compare Fig. 5I with Fig. 5A). To test
whether cmyc expression in the foregut is Wnt signaling dependent,
we overexpressed the Wnt antagonist Dkk1, starting at 21 hpf and
found a severe reduction in cmyc expression (n=25) (Fig. 5J). These
data suggest that cmyc expression is a faithful marker of endogenous
Fig. 5. Hnf1ba is required for Wnt signaling activity in the foregut endoderm. (A-C) Double antibody (myosin; blue) and in situ hybridization
staining (ventral view) of 24 hpf wild-type foregut endoderm for expression of hnf1ba (A), wnt2bb (B) and prox1 (C, arrowheads). (D) Illustration
summarizing foregut expression domains showing restricted prox1 expression within the anterior region of the hnf1ba foregut expression domain and
adjacent to the wnt2bb expression domain in the lateral plate mesoderm. Pronephric hnf1ba expression (red asterisks in A) and dorsal pancreas prox1
expression (dp) are not depicted in the illustration. (E-G) Three-dimensional rendering (lateral view) of 36 hpf foregut endoderm in wild-type (E),
Tg(hsp70l:wnt8a-GFP)w34 (F) and Tg(hsp70l:wnt8a-GFP)w34; hnf1bas430/hi2169 (G) embryos heat-shocked from 21-28 hpf at 37°C and stained for Prox1 and
myosin. Compared with wild type, heat-shocked Tg(hsp70l:wnt8a-GFP)w34 embryos show significant posterior expansion of Prox1 in the foregut
endoderm (yellow arrows; n=8/8). In Tg(hsp70l:wnt8a-GFP)w34; hnf1bas430/hi2169 embryos, only weak or no Prox1 expression is observed (yellow arrows;
n=6/6). (H-L) Double antibody and in situ hybridization staining (ventral view) of cmyc at 36 hpf in wild-type (H), Tg(hsp70l:wnt8a-GFP)w34 (I),
Tg(hsp70l:dkk1-GFP)w32 (J), hnf1bas430−/− (K) and Tg(hsp70l:dkk1-GFP)w32; hnf1bas430/hi216 (L) embryos heat-shocked from 21 to 28 hpf. cmyc expression is
restricted in the wild-type foregut endoderm (white arrowhead in H) but is expanded posteriorly in Tg(hsp70l:wnt8a-GFP)w34 (yellow arrows in I;
n=12/12) or severely reduced in Tg(hsp70l:dkk1-GFP)w32 (white arrowhead in J) embryos. cmyc expression is reduced or lost in the hnf1bas430 foregut
endoderm (white arrowhead in K; n=4/4), and only weakly induced in Tg(hsp70l:wnt8a-GFP)w34; hnf1bas430/hi2169 embryos (yellow arrow in L; n=4/4).
DEVELOPMENT
Hepatopancreas specification
Wnt signaling activity in the foregut endoderm. In hnf1bas430
homozygotes, cmyc foregut expression is markedly reduced,
suggesting that hnf1ba function is important for Wnt signaling in the
foregut endoderm (Fig. 5K; n>30). Furthermore, in hnf1bas430/hi2169
transheterozygotes, cmyc foregut expression cannot be robustly
induced by ectopic Wnt8, suggesting that hnf1ba is important for
foregut endoderm Wnt signaling activity (Fig. 5L; 100%, n=12; and
inconsistently induced in hnf1bas430 homozygotes). Because these
results demonstrate that hnf1ba is required for the expression of a
known transcriptional target of Wnt signal transduction, cmyc, we
suggest that hnf1ba functions to generate a permissive domain in
the foregut endoderm for Wnt signaling. A role for Hnf1ba in Wnt
signaling could explain the severe, yet localized, foregut defect
observed when combining the wnt2bbs404 and hnf1bas430 mutations.
In wnt2bbs404; hnf1bas430 double homozygous mutants, Wnt2bb loss
of function would be compounded by the reduced foregut Wnt
signaling that results from Hnf1ba partial loss of function. We
suggest that this synergistic reduction of Wnt signaling contributes
to the loss of hepatopancreas specification in wnt2bbs404; hnf1bas430
double mutants. It is possible that non-Wnt-related targets of
Hnf1ba also contribute to the specification of hepatopancreas
progenitors.
DISCUSSION
Our studies suggest that Hnf1ba and Wnt2bb coordinate the
specification of the progenitor population that gives rise to the liver,
ventral pancreas, gall bladder and associated ducts, but not
neighboring endodermal tissue such as the dorsal pancreas,
demonstrating a common genetic mechanism for specification of
the entire ventrally derived hepatopancreas system. Although it has
been suggested that genetic events regulating liver and pancreas
specification occur during early somitogenesis (Chung et al., 2008;
Wandzioch and Zaret, 2009), we show the Hnf1ba and Wnt
signaling interact later, only several hours prior to liver budding, to
induce hepatopancreas fate. It is well established that signals from
the mesoderm play a key role in patterning the foregut endoderm
during development (Kumar et al., 2003; Ober et al., 2006; Sneddon
et al., 2012). But it is less clear how the endoderm regulates the
reception of these signals. We provide in vivo evidence that Hnf1ba
function is required for the foregut endoderm to respond to Wnt2bb
signaling from the mesoderm.
We anticipate that this conceptual advance in understanding how
hepatopancreas progenitors are normally specified could contribute
to more efficient production of differentiated liver or pancreas cells
to potentially treat liver disease and diabetes. HNF1B+ cells are
produced in protocols designed to generate pancreatic endocrine
cells from embryonic stem cells (Kroon et al., 2008). Based on our
discoveries, we suggest that stimulating Wnt signaling in such
HNF1B+ endoderm cells would increase the efficiency of current
stem cell differentiation protocols by increasing the hepatopancreas
progenitor pool. Furthermore, an in vitro study using human
embryonic stem cells suggests that a restricted level of Wnt
signaling, at a stage when HNF1B expression is robust, can enhance
development of pancreas fate by 15-fold (Nostro et al., 2011),
supporting a positive role for Wnt signaling in human pancreas
development. This apparent functional conservation further
validates zebrafish as a reliable vertebrate model for human
pancreas development. Notably, our work suggests that there is a
narrow developmental window when positive Wnt signaling input
is required for specification of both liver and pancreas progenitors.
Because Wnt signaling outside this window may have negative
effects on pancreas specification (Murtaugh, 2008), precise
Development 140 (13)
stimulation of HNF1B+ endoderm cells by Wnt signaling in
differentiation protocols may be necessary. Future studies to
determine how progenitors specified by Hnf1b and Wnt signaling
are subsequently allocated into distinct liver, pancreas, gall bladder,
or duct fate will be important to further enhance stem cell
differentiation efforts.
Despite the discovery of MODY5 over 14 years ago, the role
HNF1B plays in pancreas development and diabetes has remained
elusive, hindered by the lack of a suitable animal model system.
Homozygous Hnf1b-null mutants exhibit broad defects that
preclude pancreas and liver morphogenesis studies. Furthermore,
heterozygous null Hnf1b mutants were not reported to exhibit
MODY5-like pancreas defects, suggesting that Hnf1b dose
regulation and/or requirements are different among vertebrates. Our
studies demonstrate the utility of a homozygous hypomorphic
hnf1ba mutant model that mimics pancreas pathologies observed in
humans with heterozygous HNF1B mutations. Interestingly,
variation in the severity of pancreas hypoplasia found in this hnf1ba
zebrafish model is also characteristic of MODY5 cases (BellannéChantelot et al., 2004). This variation is even found among patients
within the same family (Barbacci et al., 2004; Edghill et al., 2006a).
We postulate that, like homozygous hnf1bas430 zebrafish mutants,
heterozygous HNF1B mutations in individuals with MODY5 result
in a reduction of HNF1B function to a crucial threshold that is
sensitive to stochastic developmental variation (Gärtner, 1990;
Baranzini et al., 2010; Raj et al., 2010), which can be sensitive to
variations in genetic background. Using this novel MODY5 model,
we reveal that reduced Hnf1ba function can compromise
developmental β-cell numbers, and show that this reduction is not
secondary to pancreas hypoplasia defects. Furthermore, as we find
in hnf1bas430 zebrafish mutants, exocrine pancreas hypoplasia and
islet disorganization were also observed in a study of two human
fetuses harboring heterozygous HNF1B mutations (Haumaitre et
al., 2006). These common defects suggest that Hnf1b function in
pancreas development is broadly conserved among vertebrates.
However, a reduction in the number of β-cells was not reported in
that study. We suggest that, similar to the variation in pancreas
hypoplasia in both individuals with MODY5 and zebrafish
hnf1bas430 homozygotes, a decrease in embryonic β-cell numbers
in MODY5 is also variable – which may explain the variable onset
of MODY5 diabetes. In support of this idea, rare cases of neonatal
diabetes have been reported for MODY5, suggesting that β-cell
numbers may be developmentally compromised in some people
with HNF1B mutations (Edghill et al., 2006b; Beckers et al., 2007).
In addition to the previously suggested roles for Hnf1b in β-cell
insulin secretion and hepatic insulin signaling (Wang et al., 2004;
Brackenridge et al., 2006; Kornfeld et al., 2013), our findings raise
the possibility that a reduction in β-cell number may be a significant
contributing factor to the etiology of HNF1B diabetes, potentially
contributing to insulin dependence in MODY5. Furthermore, using
this hypomorphic Hnf1b mutant model, we provided definitive
evidence that hnf1ba plays a key role in ventral pancreas
specification (Haumaitre et al., 2005), upstream of ptf1a and distinct
from its roles in regulating foregut regionalization and liver
specification. By revealing specific roles for hnf1ba in β-cell and
pancreas development, our findings set the stage for future work to
explore the mechanism of these crucial functions.
Our studies revealing an unexpected coordinate role of hnf1ba
with wnt2bb in hepatopancreas specification also lays the
foundation for future research to explore how Hnf1ba, as a
transcription factor, functions to generate a Wnt permissive foregut
endoderm and whether this genetic interaction is conserved in other
DEVELOPMENT
2676 RESEARCH ARTICLE
tissues or in disease. Hnf1ba could be functioning in parallel with
Wnt signaling by independently regulating Wnt target genes or
upstream of Wnt signaling by regulating a component of the Wnt
signal transduction pathway, including co-factors. Previous
biochemical studies have suggested that Hnf1b can regulate target
genes via epigenetic modification (Barbacci et al., 2004; Verdeguer
et al., 2010). Future studies that seek to determine the precise
molecular nature of this genetic mechanism will need to investigate
these avenues of Hnf1b function. An understanding of this
interaction may also have implications for their potential interaction
in disease, as GWA studies have revealed that both HNF1B and the
Wnt effector gene TCF7L2 are strongly associated with T2D
(Frayling, 2007). With our finding that Hnf1ba is required for
expression of the direct Wnt signaling target cmyc, we suggest that
a possible consequence of HNF1B deficiency in diabetes may be
the attenuation of Wnt signaling. Consistent with this model, a
human hypoplastic MODY5 fetal pancreas was shown to have
decreased expression of a Wnt signaling component β-catenin
(Haumaitre et al., 2006).
The hypomorphic hnf1bas430 allele presented here will be a
valuable model for further investigations into the genetic
mechanisms of embryonic and post-embryonic pathologies
associated with MODY5, including maturity onset diabetes and
pancreas, liver and kidney dysfunctions. Because mouse Hnf1b-null
heterozygotes were not reported to have MODY5-like phenotypes,
we suggest that a mammalian model with a homozygous Hnf1b
hypomorphic mutation similar to hnf1bas430 may be necessary to
phenocopy MODY5 pancreas defects. This approach of using
hypomorphic alelles was shown to be necessary to model
heterozygous SOX2 mutations in humans with anophthalmiaesophageal-genital (AEG) syndrome (Que et al., 2007). Importantly,
an Hnf1b mouse model with a broad and partial reduction in Hnf1b
function would be more comparable with the MODY5 genetic
condition than would a conditional Hnf1b knockout model. Such a
mouse would be valuable to complement our findings on the
functions of Hnf1b in vertebrate hepatopancreas progenitor
specification, particularly if combined with precise Wnt signaling
inhibition.
Acknowledgements
We thank Jeremiah Gilmore for molecular biology support, Haowen Zhou,
David Nguyen, Ana Ayala and Steve Waldron for expert help with the fish, and
Pamela Itkin-Ansari, Amnon Schlegel, Elke Ober, Silvia Curado, Silvia Cereghini
and Isla Cheung for discussions and/or critical reading of the manuscript. We
thank Zhaoxia Sun, Julia Horsfield and Tatjana Piotrowski for reagents.
Funding
This work was supported in part by funds from National Institutes of Health
(NIH) [1DP2DK098092]; from Sanford Children’s Health Research Center; from
Larry L. Hillblom Foundation [2005 1G] and Juvenile Diabetes Research
Foundation (JDRF) [32002643] (to P.D.S.D.); and from NIH [DK61245 and
DK75032], JDRF and the Packard Foundation to D.Y.R.S. Deposited in PMC for
release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Author contributions
P.D.S.D. made original observations, generated initial data and hypotheses,
and submitted a manuscript while in D.Y.R.S.’s lab; P.D.S.D. and J.J.L. designed
and executed experiments, and assembled figures; K.P.G. and D.Z. contributed
to expression studies; P.D.S.D., N.Z. and T.K. contributed to mapping and
cloning of the ligers430 mutation in D.Y.R.S.’s lab; J.J.L. and L.S. genotyped
mutants; H.V. and P.D.S.D. contributed to mutagenesis screen in D.Y.R.S.’s lab;
K.S. and C.V.E.W. produced and characterized the zebrafish Pdx1 antibodies;
Y.C. provided protein structure analysis of the ligers430 mutation; P.D.S.D. and
D.Y.R.S. oversaw studies; U.S.J and R.K.H performed transcriptional analysis,
RESEARCH ARTICLE 2677
P.D.S.D., J.J.L. and D.Y.R.S. wrote the manuscript with feedback from C.V.E.W.,
K.P.G., D.Z., U.S.J., N.Z., K.S., R.K.H., T.K., L.S., H.V. and Y.C.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.090993/-/DC1
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